The present invention relates to an improved synthesis of 5-(N-[(S)-N-{N,N-bis(2-chloroethyl)amino}phenoxycarbonyl)-xcex3-glutamyl]amino)isophthalic acid (also named ZD9063P herein), a prodrug used in Antibody Directed Enzyme Prodrug Therapy (ADEPT), a targeted cytotoxic cancer therapy.
The use of cytotoxic drugs is limited by their general toxicity and a highly desirable aim is the targeted delivery of the active cytotoxic agent. A novel approach under development is the dosing of the patient with an antibody/enzyme complex which binds specifically at the site of the tumour. Subsequent treatment with a synthetic compound, the Prodrug, which bears a masked cytotoxic entity and has been specifically designed to be cleaved by the enzyme, delivers the cytotoxic agent, the Drug, at the site of the tumour.
The known synthesis of ZD9063P involved a using a nitrogen mustard, N,N-bis(2-chloroethyl)aminophenol, and being the actual cytotoxic agent and would pose very significant containment problems if used on a manufacturing scale (see U.S. Pat. No. 5,405,990; Example 46). Therefore there was a need for an improved synthetic route, particularly a route more suited to large scale manufacture.
The present invention is based on the discovery of a new, efficient and convergent route to ZD9063P and structurally related prodrugs by converting a readily available starting material to a key intermediate suitable for both chemical and enantiomeric purification. The success of the synthetic approach arose from the use of a catalyst to obtain regiospecific opening of an anhydride ring, incorporation of a reaction at an elevated pressure to avoid a solvent exchange and the development of an improved practical procedure for the deprotection of t-butyl esters.
According to one aspect of the invention there is provided a method of preparation of a compound of Formula I or an R4 deprotected derivative thereof 
wherein
R1 and R2 independently represent chloro, bromo, iodo or OSO2Me;
R3 represents COOH or a salt of carboxylic acid;
n is 1,2, 3 or 4;
R4 represents a protected form of COOH, tetrazol-5-yl or SO3H; and
in which the method comprises reacting a compound of Formula II: 
with a compound of Formula III: 
to give a compound of Formula I and optionally further comprising deprotecting R4 and optionally converting the product thus obtained into a pharmaceutically acceptable salt thereof.
R1 and R2 are preferably chloro. R3 is preferably an alphamethylbenzylamine salt of carboxylic acid. R4 is preferably a protected form of COOH, especially COOBn. Preferably n is 2 and especially a 3,5 dicarboxylic acid in protected form is preferred. Preferably the asterisked chiral carbon in Formula I has S configuration.
Preferably the reaction is performed in the presence of a highly nucleophilic but weakly basic amine. Preferably the amine is DMAP (4-dimethylaminopyridine) or 4-pyrrolidinopyridine, especially DMAP. This has the advantage of regioselective opening of the cyclic anhydride ring.
Preferably the reaction is performed at a temperature of xe2x88x9250xc2x0 to 30xc2x0, more preferably xe2x88x9240 to 0xc2x0, more preferably xe2x88x9240 to xe2x88x9210xc2x0 and especially at about xe2x88x9235 to xe2x88x9225xc2x0. Preferred temperature ranges have the advantage of giving improved yield of desired reaction products.
According to another aspect of the invention there is provided a method of preparation of a a compound of Formula II which comprises removing the tBu protecting groups from a compound of Formula IV: 
in the presence of methane sulphonic acid followed by cyclisation to the anhydride to give a compound of Formula II. This is advantageous because the de-esterification could be readily achieved by the extended reaction of trifluoroacetic acid but a significant practical problem encountered was the complete removal of the excess trifluoroacetic acid prior to the activation step. A large excess of trifluoroacetic acid is required to displace the equilibrium set up between its t-butyl ester and the corresponding t-butyl ester of the substrate. Complete reaction is normally only achieved by multiple treatments removing volatile substances by distillation, a time-consuming procedure for large scale operation. The t-butyl ester of methanesulphonic acid is unstable and so the corresponding equilibrium can be displaced by the formation of isobutylene which escapes from the system.
According to another aspect of the invention there is provided a method of preparation of a compound of Formula IV in which R1 and R2 are chloro which comprises reacting a compound of Formula V: 
with methane sulphonyl chloride in the presence of methylene chloride in a pressure vessel to give a compound of Formula IV in which R1 and R2 are chloro. This is advantageous for the following reasons. The readily available starting material reacts with methanesulphonyl chloride/diisopropylethylamine in methylene chloride solution to give an essentially quantitative yield of the corresponding dimesylate. The mesylate groups (also termed xe2x80x9cMsxe2x80x9d or xe2x80x9cmethanesulphonylxe2x80x9d herein) are displaced by the chloride ion present but the reaction is very slow even at reflux. An alternative solvent is not an attractive option as methylene chloride is the most suitable solvent for obtaining an initial solution of starting material, a necessary requisite for a high chemical conversion to the dimesylate. The solvent used must be compatible with the strongly acidic reaction conditions required for removal of the t-butyl groups as the isolation of 10 (for numerically identified compounds, see Schemes below) as a crystalline solid is not easy and it is more convenient to proceed directly with the deprotection stage. In addition, both 10 and 14 contain the nitrogen mustard system, albeit in a less activated form, and handling of such intermediates would cause concern on a production scale. The problem of the slow rate of the displacement reaction at reflux in methylene chloride solution (40xc2x0 C.) was overcome, without introducing a change to a higher boiling solvent, by operating the reaction under pressure. Complete exchange occurs after 18 hours at 75xc2x0 C. and the pressure generated in the system, about 2 BarG, is entirely consistent with operation in a standard production plant.
According to another aspect of the invention there is provided a compound of Formula I in which R1 and R2 are chloro, R3 is an alphamethylbenzylamine salt of carboxylic acid and (R4)n represents a benzyl protected 3,5-dicarboxylic acid. Preferably the compound is in crystalline form.
Another aspect of the invention comprises a compound of Formula I 
in which R1 and R2 are chloro, R3 is an alphamethylbenzylamine salt of carboxylic acid, (R4)n represents a benzyl protected 3,5-dicarboxylic acid and the asterisked chiral carbon in Formula I has S configuration. Preferably the compound is in crystalline form wherein said alphamethylbenzylamine is in enantiomerically pure (R)- or (S)-configuration.
The crystalline form of this compound gives the advantage of ease of handling during manufacture, particularly at large scale.
Explanation of the discovery of the advantageous synthetic route is set out below. A molecule 8, possessing the same basic aryl urethane derivative of (S)-glutamic acid as ZD9063P, was available.
(Scheme 1)
The two key transformations required are the conversion of the hydroxyl groups to chloro groups and the regioselective coupling of the dibenzyl ester of 5-aminoisophthalic acid with the glutamic acid residue giving a compound 9 which can be converted to ZD9063P by catalytic hydrogenation. The key decision is the order in which the chlorination reaction and the introduction of the dibenzyl ester of 5-aminoisophthalic acid moiety are carried out.
(Scheme 2)
Initially, the partial hydrolyses of 8 and of its dichloro-analogue 10 were investigated to provide differentially protected derivatives of glutamic acid. Trifluoroacetic acid selectively cleaved the less hindered ester group but it was not possible to obtain solution yields of the desired regioisomer of greater than about 50% because further de-esterification continually occurred.i 
The physical characteristics of the chlorinated species as well as the desire to avoid the conversion of the hydroxyl group to the chloro group at a late stage in the synthesis suggested that a dichloro derivative should be chosen as the key starting material for coupling with the dibenzyl ester of 5-aminoisophthalic acid. In addition, approaches based on the opening of the corresponding cyclic anhydride were considered to be more selective than activation of the acyclic system with, for example, a chloroformate ester.
Control of the regioselective opening of anhydrides of glutamic acid derivatives by methanol in the presence of triethylamine containing varying amounts of DMAP has been described. The ratio of regioisomers is reversed from about 1:7 to about 7:1 xcex1/xcex3 by the addition of DMAP.ii 
Regioselective opening of the anhydrides of phthaloyl derivatives of glutamic acid with amines has been described leading to the xcex3-isomer and specific reference was made to the opening of the anhydrides of urethane derivatives of the anhydride of glutamic acid with ammonia leading to predominantly the xcex1-isomer.iii The regioselectivity of reaction with amines has also been controlled in urethane derivatives of glutamic and aspartic acid anhydrides by choice of reaction solvent.iv v vi No examples in which DMAP affected the regioselectivity of nucleophilic attack by amines on derivatives of the cyclic anhydrides of glutamic acid have been found.
The invention will now be illustrated by the following non-limiting Examples in which:
Scheme 1 shows synthesis of compound 8 in which a=TMS-Cl, b=4-nitrophenol chloroformate, c=esterification, d=reduction, e=hydroxyethylation
Scheme 2 shows options for key transformations in which the bold arrows show the route developed with key intermediates and the dotted arrows show alternative route options with possible intermediates
Scheme 3 shows a model experiment
Scheme 4 shows a new synthetic route to ZD9063P in which a=MsCl/Hunig""s base, b=heat Reagents were purchased from standard suppliers.
NMR spectra were run at 270 MHz (proton) and at 67.7 MHz (carbon) in d6-DMSO or d6-DMSO/TFA solution and are reported in parts per million down field from internal TMS. The signals assigned to TFA (159.0, q, J=60.9 Hz, 115.3, q, J=440 Hz) are omitted from the description of the 13C spectrum for each compound.
HPLC analyses were conducted using a HiChrome(trademark) RPB column, solvent system acetonitrile/water/TFA 640/360/1 (v/v/v), flow rate 1 or 2 mL/min and detection at 254xcex